Enzymes produced by koji molds have been used in a diversity of industries.
For example, various enzymes produced by koji molds have been utilized in producing soy sauce which is a traditional food in Japan. To produce soy sauce, a koji mold is grown in soybeans and wheat, which are the starting materials, and allowed to produce various enzymes. These enzymes produced by the koji mold decompose proteins, saccharides, lipids and so on and promote lactic acid fermentation and yeast fermentation in the subsequent steps. When the koji mold produces a large amount of enzymes which decompose the starting materials during the process, the utilization efficiency of the starting materials and compression level can be improved to largely enhance the productivity. Additionally, since a sufficient amount of substrates to the lactic acid fermentation and yeast fermentation is provided, the fermentations are appropriately conducted. Thus, the qualities of soy sauce are highly improved.
Accordingly, it is very important from an industrial standpoint to breed koji molds having high enzyme productivity. For this purpose, breedings have been energetically conducted so far. Since the entire genome sequence of Aspergillus oryzae RIB40 was identified (Non-Patent Document 1), this strain becomes applicable to breeding.
Methods for breeding koji molds with a purpose of high enzyme productivity are roughly classified into the mutation method and the genetic recombination method.
The genetic recombination method comprises introducing a target gene into a koji mold with the use of transformation to breed the transformant. Usually, genes of 5 to 6 kb in size are introduced by the method. Introduction of a gene of 10 kb or larger is very difficult to succeed because of a considerable decrease in the transformation efficiency. Additionally, the region to be introduced includes the promoter region, structural gene region or terminator region of the target gene or, in some cases, a gene which can serve as a marker in screening. Thus, it is difficult to introduce multiple genes in a fragment of 10 kb or smaller. Therefore, the genetic recombination method is effective in an industrial field such as enzyme production where it is merely required to highly produce a single enzyme alone while in an industrial field such as food manufacturing where a plural enzymes for decomposing starting materials should be highly produced simultaneously, the genetic recombination method is not effective. In soy sauce manufacturing cited as an example of food manufacturing, it is necessary to produce a large amount of various kinds of enzymes including enzymes decomposing various starting materials for enhancing the yield, various enzymes for enhancing the level of compression, enzymes for enriching the body taste and so on. To highly produce these various kinds of enzymes at the same time by the genetic recombination method, transformation should be repeatedly carried out. For this purpose, it is necessary to construct a system whereby a marker to be used in screening a transformant can be recycled. However, it is extremely difficult to construct such a system. Even supposing that transformation is repeatedly carried out and thus genes of a large variety of enzymes are inserted into genomes, the inserted genes cannot always sufficiently function because of the problems relating to gene loci and expression control systems. Moreover, since the enzyme production mechanisms of koji molds still remain unknown in many points, there is no guarantee that the productivity of a target enzyme can be enhanced merely by inserting a gene.
Additionally, foods manufacturing using gene recombination technology is still unacceptable in the Japanese market. Thus, there are some problems in the application of the genetic recombination method to food manufacturing in practice. Therefore, breedings have been conducted by the mutation method using, for example, ultraviolet light irradiation. However, the method causes an associated mutation other than the target mutation. In the case, therefore, even though a mutant having an enhanced productivity of the desired enzyme can be obtained, there frequently arise some problems such as a slow growth rate, a decrease in the productivity of another enzyme, etc.
Furthermore, a koji mold mutant is genetically unstable in many cases. Namely, it is frequently observed that the property of a mutant becomes identical with that of the parent strain thereof as the mutant grows (hereinafter this phenomenon will be called “reverse mutation”), which brings about troubles in the industrial application.
Accordingly, although a koji mold which highly produces a variety of enzymes simultaneously has been required in industries such as the food industry with a need for various kinds of enzymes at the same time, there are problems as discussed above.
Examples of known methods for breeding a koji mold which highly produce enzymes with the use of the mutation method are as follows: a method which comprises treating Aspergillus oryzae RIB128 with N-methyl-N′-nitro-N-nitrosoguanidine (hereinafter referred to as NTG) to obtain a strain having a high phytase activity and reduce the amount of phytin in sake and increase the amount of inositol which is a biologically active substance (Patent Document 1); a method which comprises treating Aspergillus oryzae AJ117281 with NTG to obtain a mutant having a high protease activity and produce a nitrogen-rich protein hydrolysate (Patent Document 2); a method which comprises treating Aspergillus oryzae AJ117290 (FERMP-14259) with NTG to obtain a mutant having a high glutaminase activity and produce a protein hydrolysate having a high glutamic acid content (Patent Document 3); and so on. With respect to the induction of mutation, examples of agent for the induction of mutation not only include NTG as described above but also other chemicals commonly used such as hydroxylamine, ethylmethylsulfonic acid and the like or irradiation with ultraviolet light, radial ray, X-ray and the like.
Other examples of known breedings using the mutation method include: a method which comprises mutagenizing Aspergillus oryzae O-1013 (FERM P-16528) with the use of NTG to obtain a mutant which highly produces deferriferrichrysin (Patent Document 4); a method which comprises treating Aspergillus sojae with ultraviolet light to obtain a white koji mutant having white conidia and obtain miso with a good color hue (Patent Document 5); a method which comprises mutagenizing Aspergillus oryzae with the use of, for example, X-ray to obtain a strain having a reduced isovaleraldehyde productivity for preventing the occurrence of a stuffy smell that is a deteriorated odor of sake (Patent Document 6); a method which comprises irradiating red koji mold (Monascus) with heavy ion beam to obtain a koji mold strain which highly producing monacolin K which is a cholesterol-lowering substance (Patent Document 7); a method which comprises irradiating a microorganism with iron ion beam imparting linear energy to transfer insertion mutation and deletion mutation of about 1.2 kb (Patent Document 8); an example wherein a black koji mold mutant was constructed using heavy ion beam (Non-Patent Document 2); and so on.